|
| Status |
Public on Jun 09, 2016 |
| Title |
BrdU IP in cdc25-22 synchronized delta taz1 cells 120 min |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
cdc25-22 delta taz1_synchronized_IP DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: cdc25-22 ∆taz1 cells
molecule subtype: BrdU immunoprecipitated DNA
|
| Treatment protocol |
Cells were collected at time intervals corresponding to maximum septation index and fixed with 0.1% sodium azide.
|
| Growth protocol |
Cdc25-22 cells carrying thymidine kinase expression module Pnmt1-TK and nucleoside transport module Padh1-hENT were grown in minimal media at 26˚C and arrested at the G2/M block by raising temperature to 37˚C for 4h and 15 min. Cells were released from block by reducing temperature to 26˚C, and cultures were supplemented at this time by 10mM hydroxyurea and 200μΜ 5-bromo-2'-deoxyuridine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The DNA was phenol-extracted from fixed cells by bead-beating with glass beads, sheared by sonication to 500-1000bp fragments and immunoprecipitated with anti-BrdU antibody (BD Pharmigen) immobilized to Dynal anti-mouse magnetic beads (Invitrogen).
|
| Label |
Cy5
|
| Label protocol |
Immunoprecipitated and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (IP DNA) or Cy3 (whole-cell extract DNA).
|
| |
|
| Channel 2 |
| Source name |
cdc25-22 ∆taz1_synchronized_whole-cell extract
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: cdc25-22 delta taz1 cells
molecule subtype: Whole-cell extract DNA
|
| Treatment protocol |
Cells were collected at time intervals corresponding to maximum septation index and fixed with 0.1% sodium azide.
|
| Growth protocol |
Cdc25-22 cells carrying thymidine kinase expression module Pnmt1-TK and nucleoside transport module Padh1-hENT were grown in minimal media at 26˚C and arrested at the G2/M block by raising temperature to 37˚C for 4h and 15 min. Cells were released from block by reducing temperature to 26˚C, and cultures were supplemented at this time by 10mM hydroxyurea and 200μΜ 5-bromo-2'-deoxyuridine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The DNA was phenol-extracted from fixed cells by bead-beating with glass beads, sheared by sonication to 500-1000bp fragments and immunoprecipitated with anti-BrdU antibody (BD Pharmigen) immobilized to Dynal anti-mouse magnetic beads (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
Immunoprecipitated and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (IP DNA) or Cy3 (whole-cell extract DNA).
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy5-labeled ChIP DNA and Cy3-labeled whole-cell extract DNA were mixed and combined with human Cot1 DNA, Agilent Blocking Agent and Agilent Hybridization buffer, and hybridized to high-density microarrays in Agilent SureHyb hybridization chamber for 24 hours at 65˚C, 10 rpm. After hybridization, slides were washed according to Agilent protocol.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner.
|
| Description |
BrdU IP ∆taz1 120min V3 series1
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software (CHIP_1105_Oct12 ). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization.
Enrichment values were calculated as a ratio of Cy5 processed signal/ Cy3 processed signal.
|
| |
|
| Submission date |
Mar 02, 2016 |
| Last update date |
Jun 10, 2016 |
| Contact name |
Shiv Grewal |
| Phone |
2407607553
|
| Organization name |
NCI
|
| Department |
LBMB
|
| Lab |
Shiv Grewal
|
| Street address |
NCI bldg 37 Rm 6068 9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL6503 |
| Series (2) |
| GSE78819 |
Taz1-Shelterin promotes facultative heterochromatin assembly at chromosome-internal sites containing late replication origins [BrdU] |
| GSE78823 |
Taz1-Shelterin promotes facultative heterochromatin assembly at chromosome-internal sites containing late replication origins |
|
